Using highly sensitive CCD cameras, single fluorescent molecules can be detected and imaged as fluorescent spots (in the absence of background fluorescence). Fitting a single fluorophore such that its center can be located to nanometer accuracy has enabled “super resolution” microscopy, i.e., resolution much better than 200-300 nm accuracy. Using photoswitchable fluorophores (via PALM [1] or STORM [2, 3]) or by forcing dyes into long-lived dark states (via dSTORM [4]), individual fluorophores are visible, even when fluorophore density is high. Because the individual fluorophores are visible and can be fit to two-dimensional Gaussian functions, the positions of the fluorophores can be localized to nanometer accuracy (5, 6). The positions of the individual fluorophores can then be used to draw a composite super-resolution image.
Here we show that it is unnecessary to see single spots to achieve super-resolution imaging. Fluorophores stochastically photobleach when exposed to laser excitation. Resulting quantized drops in fluorescence intensity can be localized, even with background, to create super-resolution images from standard photobleaching movies. This is achieved by subtracting post-photobleaching images from pre-photobleaching images. In addition, we use frame averaging and weighted two-dimensional Gaussian fitting to reduce the effects of shot noise that are inherent in the higher fluorescent background. In an analogous way, we can also localize fluorophores that blink, transitioning from dark to bright states, and vice versa. This technique thus presents a much simpler way to create super resolution images. We are calling our technique “photobleaching and intermittency localization microscopy,” or PhILM for short.